NMR (Steps, Pros, Cons)

1) Steps: purify the protein, dissolve the protein, collect NMR data, assign NMR signals, calculate structure. 2) Pros: no need to crystallize the protein, can see many hydrogens, can show which regions are flexible. 3) Cons: difficult for insoluble proteins, flexible proteins, works best with small proteins.

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Electron Tomography (Steps, Pros, Cons)

1) Steps: purify the protein, freeze the protein, collect electron density tomograph data, average the images to get high-reliability volumes, calculate the structure. 2) Pros: no need to crystallize the protein, low density regions are frequently lost 3) Cons: freezing proteins causes complications, works best with large proteins with known subunit crystal structures.

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Homology Modeling

Does not determine the true structure. Generates a structural hypothesis. Useful due to the rise in the amount of genomes sequenced. Takes a little time.

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Why do high resolution studies (2)

1) Exceptionally high quality structure information 2) Can greatly increase the understanding of a protein's function

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Why not do high-resolution studies? (3)

1) Potentially time consuming2) Technically challenging (you need a specialist)3) enzymatic function does not require a structure to answer effectively.

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Why do a low-resolution study? (2)

1) Confirm that your protein of interest still has structure (after manipulation in the lab) 2) Significantly faster.

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Why do a modeling study?

1) Because you need structural data, and there are high-similarity family members available and you lack the time or expertise to do a high-resolution study, or are working with a difficult protein.

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What are the five steps to get a purified protein?

1) Make a crude extract 2) separate the protein components (separate based on charge, size, solubility, thermal stability, affinity for a ligand, affinity for a ligand, hydrophobicity 3) Determine the purity of the protein. 4) Measure the total amount of protein 5) Calculate purification factor and yield.

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How do you separate a protein on the basis of charge?

1) Ion-exchange chromatography 2) Exploits differences in the sign and magnitude of the net electric charges of proteins at a given pH

Absorbance at 280 (Method, Pros, Cons)

1) Used to measure the amount of protein in a sample. Absorbance at 280 nm- Aromatic side chains (tyrosine & tryptophan) absorb UV light. 2) Pros: fast, easy, quantitative/ extremely accurate if a single protein of high purity is measured, since the ratio of Tyr/Trp to mass is known. 3) Cons: Not all proteins have equal amounts of Tyr/Trp. Thus very inaccurate for bulk proteins/Non-protein compounds absorb at 280 nm

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Pierce/Bradford Assay (Method, Pros, Cons)

1) Used to measure the total amount of protein. positvely charged amino acids bind to a dye, Coomassie G-250, and once bound, absorb light at 595 nm. 2) Pros: Most proteins bind to Coomassie-G-250 evenly thus high accuracy for bulk proteins. Few non protein compounds absorbs at 595 nm. 3) Cons: Takes longer than simply measuring absorbance, and must measure standards and replicate samples for accuracy.

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Purity calculation

Purity = Amount of the protein of interest/Amount of total protein

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Yield calculation

Yield = final amount of protein/initial amount of protein

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What two methods are used to identify unknown proteins?

MALDI MS and ESI MS

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What are the advantages/disadvantages of Mass Spec?

Advantages: Can be used w/ small amount of proteins and mixed samples. Provides sequence information Disadvantages: fragmentation processes leave gaps in the sequences

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Circular Dichromism (CD)

Measurements of the differences in absorption of left handed and right handed polarized light